Improvements in large-scale production of Tobacco Etch Virus protease.

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-12-14 DOI:10.1016/j.pep.2024.106648

Simon Messing, Kirsten Barnhart, Matthew Drew, Natalie Granato-Guerrero, Carissa Grose, Brianna Higgins, Min Hong, Jenna Hull, Shelley Perkins, Ivy Poon, Nitya Ramakrishnan, Amanda Seabolt, Troy Taylor, Vanessa E Wall, Nicholas Wright, William Gillette, Dominic Esposito

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引用次数: 0

Abstract

Tobacco-etch-virus (TEV) protease is the workhorse of many laboratories in which protein expression is the linchpin of downstream experiments. TEV protease is remarkable in its sequence specificity as the cleavage sequence rarely appears in higher organisms and its ability to cleave fusion tag proteins from proteins of interest. Herein we report work done on large-scale production of TEV protease using different promotors, media, fusion tags, and expression platforms. During our work we detected post-translational modification (gluconoylation and phosphogluconoylation) of TEV protease and the subsequent effects this has on the purity of the protein. Subsequently we made our pgl plus bacteria that negates these modifications and their effects. We also introduce a GFP-based assay for measurement of activity and ultimately a new set of protocols for producing 400-500 mg/L TEV protease.

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烟草蚀刻病毒蛋白酶大规模生产的改进。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.